The key safety concern with chimeric antigen receptors is
their inability to distinguish whether healthy or malignant cells are
expressing a target antigen. A group at the Memorial Sloan Kettering Cancer Center thinks
it has solved the problem with T cells expressing two antigen-specific
receptors to distinguish on-target cancer cells from off-target normal cells.1

Chimeric
antigen receptor (CAR) T cells currently in the clinic are made up of an
antigen-specific single-chain variable fragment (scFv) fused to T cell-activating
and -co-stimulatory domains. The CAR is designed to bind to an extracellular,
tumor-associated antigen, and the stimulatory domains cause T cells to activate
and proliferate, thus eliminating tumor cells.

The
MSKCC team went a step further by including in their T cell-based therapeutics
another CAR-an inhibitory CAR (iCAR) that fused an scFv to a T cell inhibitory
signaling domain (see"Putting
the brakes or gas on T cell function").

As
proof of concept, the group focused on two antigens: CD19 and prostate-specific membrane antigen (PSMA; FOLH1; GCPII). CD19 is overexpressed on
malignant B cells but also is found on normal B cells. PSMA is
overexpressed on metastatic prostate cancers but also is found on normal
kidney, liver and colon cells, and brain astrocytes.

In
the standard CAR, the researchers used T cell-signaling domains from CD3z and CD28 to activate and amplify T cell
function. Both domains are common components of most CARs in the clinic.

Similar
results were seen with T cells expressing a CD19-specific CAR plus the
PSMA-specific iCAR in cell culture and mice. T cells with combined CAR and iCAR
expression eliminated on-target
CD19+/PSMA- cells but spared
CD19+/PSMA+ cells, indicating
that iCARs also put the brakes on CAR-based T cell function.

The
group also showed that the strategy could distinguish mixed on-target and
off-target cells. To do so, the researchers made a coculture of green
fluorescent, on-target CD19+/PSMA- artificial
antigen-presenting cells and red fluorescent, off-target CD19+/PSMA+artificial
antigen-presenting cells.

Time-lapse
microscopy showed that green on-target cells and red off-target cells were
eliminated at a similar rate by T cells expressing a CD19-specific CAR. In
contrast, T cells expressing the CD19-specific CAR plus the PSMA-specific iCAR
eliminated green cells and spared red cells. After 38 hours there was about an
85% decrease in on-target cells and a 10% decrease in off-target cells.

Together,
these results suggest that the iCAR T cell system can selectively inhibit T
cells from engaging in off-target effects while still allowing them to
eliminate on-target cells.

Results
were published in Science Translational Medicine. The team was led by
Michel Sadelain, director of MSKCC's Center for Cell Engineering.

"Few
antigens are completely specific to tumor tissue, so broadening the
applicability of CAR therapy will be dependent upon finding ways to target
tumor antigens that are also expressed to a degree on healthy tissue," noted
Michael Gladstone, an associate in the life sciences group at Atlas Venture. "This new work is an excellent
indication that iCAR strategies may be a viable way to expand the tumor
antigens that may safely be targeted with highly potent CAR therapy."

Stewart
Abbot, executive director of integrative research research at Celgene Cellular
Therapeutics, a subsidiary of Celgene Corp., said that the strategy is an elegant first
step to provide a molecularly encoded control for
T cell-based therapeutics.

"The
inclusion of inhibitory signaling into engineered T cells provides the
possibility to limit potential normal tissue destruction without entirely
ablating the therapeutic agent," he said.

Map for the
iCAR

Sadelain told SciBX that his goal is
to pursue iCARs in the solid tumor setting, which contrasts with conventional
CARs that are in development for blood cancers.

"We
are interested in first taking our system into solid tumors, where there are no
targets with a profile as attractive as that of CD19," he said. "We
will not apply the iCAR approach to the CD19 setting."

Sadelain's laboratory was also part of a team that
designed T cells expressing two different receptors that ramped up T cell
function when encountering on-target cells while sparing off-target cells.
Results were published early in 2013 (ref. 2; seeBox 1, "Revving up T cells").

CD19-specific, CAR-based T cell therapies eliminate
CD19-expressing B cell malignancies and have shown dramatic results in the
clinic. The side effect of elimination of healthy B cells is considered a
reasonable trade-off even though some patients may need to undergo a lifetime
of immunoglobulin replacement therapy.

Sadelain said that another application of the iCARs is in
graft-versus-host disease (GvHD). He said that Marcel van den Brink and his lab
are working together to design iCARs to prevent GvHD during bone marrow
transplant-the iCARs block the attack of donor immune cells on the recipient's
normal tissue while allowing attack on the recipient's cancer cells.

van den Brink is the head of hematologic oncology at
MSKCC and specializes in allogeneic blood stem cell transplantation for adult
patients with cancer.

Regardless
of indication, Gladstone said, "there are still limitations with
identifying and selecting iCAR antigens that are reliably and sufficiently
robustly expressed on normal tissues but not on tumor tissue. Another potential
challenge is defining the CAR cell product."

For each antigen combination, absolute and relative levels
of CAR and iCAR expression on the T cells will have to be determined, as well
as how stable the CAR and iCAR expression is in vivo over time.

"Tumor antigens can be quite heterogeneous between
patients and even within a given patient-both on tumor and on healthy tissue-so
it is critical to understand whether variation of CAR and/or iCAR expression or
antigen density disrupts
the safety and efficacy margins exemplified in their study," said
Gladstone.

Abbot saw at least two other potential limitations. "The
current proof-of-concept studies are well conducted, but the iCAR targets may
be expressed at higher levels than would be expected in normal tissues,"
he said. "The inhibitory potential of the iCARs when engaging
physiologically relevant concentrations of their target antigens remains to be
defined."

He added, "Additionally, the effect of chronic
co-stimulation of iCAR and CAR remains to be tested. The authors themselves
point out that there may be the possibility for some-arguably the majority-of
the T cells expressing CAR plus iCAR to become energized or exhausted over
time."

Dario Campana, a professor of pediatrics at the National University of Singapore,
thought that the main application would be in solid tumors and the immediate
next steps should be testing the T cell strategies in a setting that is a
better proxy for actual malignancies. Specifically, he said, the group should
focus on two targets that are differentially expressed on human cancer and
normal cells.

"While this is a very elegant and successful
demonstration of a proof-of-concept iCAR study, to my knowledge there are no
such tumors that coexpress PSMA and CD19," said Campana. "The authors
mention other targetable antigen combinations, but whether these are truly
differentially expressed in a substantial number of tumors or if they can
produce similar effects remains to be seen."

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